U.S. patent application number 10/684268 was filed with the patent office on 2004-07-15 for methods and systems for detecting mhc class i and ii binding peptides.
This patent application is currently assigned to Beckman Coulter, Inc.. Invention is credited to Monseaux, Sylvain, Montero-Julian, Felix A..
Application Number | 20040137537 10/684268 |
Document ID | / |
Family ID | 32068790 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040137537 |
Kind Code |
A1 |
Montero-Julian, Felix A. ;
et al. |
July 15, 2004 |
Methods and systems for detecting MHC class I and II binding
peptides
Abstract
The invention is based on the discovery that MHC class I and
class II monomers immobilized to a solid surface are still capable
of forming complexes with suitable MHC-binding peptides. Methods
for detecting peptide binding to HLA monomers, and methods for
measuring the relative degree of binding between two MHC-binding
peptides as well as a method of measuring the rate of dissociation
of peptides from MHC complexes are provided. The present invention
also provides systems and kits useful for conducting the methods of
the invention.
Inventors: |
Montero-Julian, Felix A.;
(Marseille, FR) ; Monseaux, Sylvain; (Marseille,
FR) |
Correspondence
Address: |
GRAY CARY WARE & FREIDENRICH LLP
4365 EXECUTIVE DRIVE
SUITE 1100
SAN DIEGO
CA
92121-2133
US
|
Assignee: |
Beckman Coulter, Inc.
Fullerton
CA
|
Family ID: |
32068790 |
Appl. No.: |
10/684268 |
Filed: |
October 10, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10684268 |
Oct 10, 2003 |
|
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10269473 |
Oct 11, 2002 |
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Current U.S.
Class: |
435/7.5 |
Current CPC
Class: |
G01N 33/6854 20130101;
G01N 33/56977 20130101; B01J 2219/005 20130101; B01J 2219/00637
20130101; G01N 2458/00 20130101; B01J 2219/00454 20130101; G01N
2333/70539 20130101; B01J 19/0046 20130101; B01J 2219/0061
20130101; B01J 2219/00605 20130101; B01J 2219/00315 20130101; B01J
2219/0074 20130101; B01J 2219/0063 20130101; B01J 2219/00621
20130101; B01J 2219/00725 20130101; G01N 33/54306 20130101 |
Class at
Publication: |
435/007.5 |
International
Class: |
G01N 033/53 |
Claims
What is claimed is:
1. A system comprising a solid surface, wherein the surface has
attached thereto one or more MHC monomer or modified MHC monomer
wherein the monomer incorporates from solution a suitable
MHC-binding peptide.
2. The system of claim 1, wherein the monomer is MHC class I and
the monomer incorporates the MHC-binding peptide under
reconstituting conditions.
3. The system of claim 1, wherein the solid surface is a bead.
4. The system of claim 1, wherein the solid surface is in a
microtiter plate.
5. The system of claim 1, wherein the solid surface is suitable for
screening in a high throughput system.
6. The system of claim 1, wherein attachment of the monomer to the
solid surface is reversible.
7. The system of claim 1, wherein attachment of the monomer to the
solid surface is cleavable.
8. The system of claim 1, wherein the solid surface is coated with
a first binding ligand and the C-terminal end of the monomer is
provided with a second binding ligand, wherein the first ligand
binds specifically with the second ligand.
9. The system of claim 7, wherein the first binding ligand is
selected from avidin, streptavidin, neutravidin, StrepTactin and
monomeric avidin and the second binding ligand is biotin.
10. The system of claim 8, wherein the second binding ligand is
attached to the monomer via a C-terminal end.
11. The system of claim 1, wherein the monomer is an MHC class I
monomer and the monomer denatures under denaturing conditions and
reconstitutes to incorporate the MHC-binding peptide under
renaturing conditions.
12. The system of claim 11, wherein the denaturing conditions
comprise a pH of about 2 to about 4.
13. The system of claim 1, wherein the system further comprises an
anti-MHC antibody that binds specifically to a conformational
epitope that is present in the reconstituted monomer and absent in
the denatured monomer.
14. The system of claim 1, wherein the reconstituting conditions
include a pH of from about 7 to about 8.5.
15. The system of claim 1, wherein the system further comprises
beta-2 microglobulin.
16. The system of claim 14, wherein the monomer is HLA class I.
17. The system of claim 15 further comprising an anti-MHC class I
monoclonal antibody, wherein the monoclonal antibody specifically
binds to a reconstituted monomer and does not bind to a denatured
monomer.
18. The system of claim 17, wherein the system further comprises
beta-2 microglobulin and a suitable HLA-binding peptide of from
about 8 to about 12 amino acids; wherein a reconstituted monomer
binds to the beta-2 microglobulin and the suitable peptide under
reconstituting conditions.
19. The system of claim 17, wherein the monoclonal antibody is
produced by hybridoma B9.12.1
20. The system of claim 1, wherein the monomer is HLA class II.
21. The system of claim 20 further comprising a monoclonal antibody
that distinguishes between a monomer bound to a MHC-binding peptide
and a monomer that lacks a MHC-binding peptide.
22. The system of claim 21, wherein the monomer is HLA class II and
the system further comprises a suitable HLA-binding peptide of from
about 10 to about 30 amino acids; wherein a reconstituted monomer
binds to the suitable peptide under reconstituting conditions.
23. The system of claim 1, 17, or 22 wherein the solid surface
coated with the monomers is in a dried form.
24. A kit comprising the system of claim 1, 17 or 22.
25. The kit of claim 23 further comprising an instruction.
26. The kit of claim 24 further comprising a control peptide to
which the MHC monomer binds in a reconstituted form.
27. A method for determining binding between a MHC class I monomer
or modified MHC class I monomer and a putative MHC-binding peptide
therefor, said method comprising: incubating under reconstituting
conditions a solid surface having attached thereto a plurality of
MHC monomers or modified MHC monomers in the presence and absence
of the putative MHC-binding peptide, wherein the monomers have been
denatured and reconstitute to form a complex containing a suitable
MHC-binding peptide under reconstituting conditions, and
determining binding to the MHC monomers after contact therewith of
a monoclonal antibody that binds to the MHC complex but does not
bind to dissociated components of the MHC complex, which binding of
the antibody indicates binding of the monomers with the putative
MHC-binding peptide.
28. The method of claim 27, wherein the denaturing conditions
include a pH in the range from about 2 to about 4.
29. The method of claim 27, further comprising separately
incubating the monomers with a standard MHC-binding peptide for the
monomers under the reconstituting conditions in the presence of the
monoclonal antibody, and wherein the determining includes comparing
binding of the antibody caused by the standard peptide to the
binding of the antibody caused by the putative MHC-binding
peptide.
30. The method of claim 29, wherein the monomers are HLA class I,
the monoclonal antibody is an anti-MHC-class I antibody, and the
reconstituting conditions include the presence of sufficient beta-2
microglobulin for reconstitution of the monomers.
31. The method of claim 30, wherein the monomers are HLA subclass
A, B or C.
32. A method for determining binding between a MHC class II monomer
or modified MHC class II monomer and a putative MHC-binding peptide
therefor, said method comprising: incubating under suitable peptide
loading conditions a solid surface having attached thereto a
plurality of MHC monomers or modified MHC monomers in the presence
and absence of the putative MHC-binding peptide to form a complex
containing a suitable MHC-binding peptide, and determining binding
of the MHC monomers with the putative MHC-binding peptide.
33. The method of claim 32, wherein the monomers are HLA class II
and the determining comprising contacting the complex with a
monoclonal antibody that determines the binding of the MHC monomers
with the putative MHC-binding peptide.
34. The method of claim 32 wherein the antibody distinguishes
between a monomer that is bound to an MHC-binding peptide and a
monomer that is not bound to an MHC-binding peptide.
35. The method of claim 32, wherein the suitable peptide loading
conditions include a pH in the range from about 4 to about 8.
36. The method of claim 32, further comprising separately
incubating the monomers with a standard MHC-binding peptide for the
monomers under the suitable conditions, and wherein the determining
includes comparing binding of the standard peptide to the binding
caused by the putative MHC-binding peptide.
37. The method of claim 32, wherein the monomers are HLA subclass
D, DR, DP or DQ.
38. The method of claim 27 or 33, wherein the monoclonal antibody
is provided with a detectable label and the determining includes
detecting the detectable label.
39. The method of claim 38, wherein the detectable label is
peroxidase.
40. The method of claim 38, wherein the detectable label is a
secondary antibody that specifically binds to the monoclonal
antibody.
41. The method of claim 40, wherein the detectable label is
fluorescent.
42. The method of claim 27 or 33, wherein the solid surface is the
wells of a microtiter plate or beads and the determining includes
reading fluorescence with a fluorometer.
43. The method of claim 42, wherein the detecting further comprises
detecting the fluorescence using high throughput scanning.
44. The method of claim 27 or 32, wherein the solid surface is
coated with avidin and the monomers are biotinylated to attach to
the solid surface.
45. The method of claim 27 or 21, wherein attachment of the
monomers to the solid surface is reversible.
46. The method of claim 27 or 32, wherein attachment of the
monomers to the solid surface is cleavable.
47. A method for determining the degree of binding affinity of an
MHC class I monomer or modified MHC class I monomer for a putative
MHC-binding peptide therefor, said method comprising: incubating at
least one denatured MHC monomer or modified MHC monomer attached to
a solid surface with the putative MHC-binding peptide and a
monoclonal antibody that specifically binds to a conformational
epitope in a first complex containing a corresponding reconstituted
MHC monomer and does not bind to any dissociated component of the
MHC complex, wherein the incubation is under reconstituting
conditions; and comparing binding of the monoclonal antibody to the
MHC complex that contains the putative MHC-binding peptide with
binding of the monoclonal antibody to a corresponding complex
containing the monomer and a known MHC-binding peptide, wherein a
difference in the bindings indicates the relative degree of binding
affinity of the reconstituted monomer for the putative MHC-binding
peptide.
48. The method of claim 47, wherein the reconstituting conditions
include a temperature in the range from about 4.degree. C. to about
37.degree. C.
49. The method of claim 47, wherein the reconstituting conditions
include a temperature in the range from about 4.degree. C. to about
8.degree. C.
50. The method of claim 47, wherein the reconstituting conditions
include a pH in the range from about 7 to about 8.5.
51. The method of claim 47, wherein the reconstituting conditions
include the presence of a suitable reconstitution buffer.
52. The method of claim 47, wherein the monomers further bind with
beta-2 microglobulin in reconstituting conditions and the
monoclonal antibody is an anti-MHC-class I monoclonal antibody.
53. The method of claim 51, wherein the monomers are selected from
HLA-A, HLA-B, and HLA-C.
54. The method of claim 53, wherein the monoclonal antibody is
produced by hybridoma B9.12.1.
55. A method for determining the degree of binding affinity of an
MHC class II monomer or modified MHC class II monomer for a
putative MHC-binding peptide therefor, said method comprising:
incubating under suitable binding conditions at least one MHC class
II monomer or modified MHC class II monomer attached to a solid
surface with the putative MHC-binding peptide and a monoclonal
antibody that specifically binds to an epitope in a first complex
containing the monomer and does not bind to any dissociated
component of the MHC complex; and comparing binding of the
monoclonal antibody to the MHC complex that contains the putative
MHC-binding peptide with binding of the monoclonal antibody to a
corresponding complex containing the monomer and a known
MHC-binding peptide, wherein a difference in the bindings indicates
the relative degree of binding affinity of the monomer for the
putative MHC-binding peptide.
56. The method of claim 55, wherein the binding conditions include
a pH in the range from about 4 to about 8.
57. The method of claim 47 or 55, wherein the incubating is for a
period of from about 12 hours to about 48 hours.
58. The method of claim 47 or 55, wherein the antibody is provided
with a detectable label and wherein the comparison of binding
comprises detecting a difference in the respective signals produced
by the detectable label resulting from binding of the antibody
thereto.
59. The method of claim 58, wherein the antibody is labeled with a
fluorescent label and the comparison of binding comprises detecting
a difference in the respective fluorescence resulting from binding
of the antibody thereto.
60. The method of claim 59, wherein the fluorescent label is
fluorescein isothiocyanate (FITC).
61. The method of claim 47 or 55, wherein the solid surface is the
wells of a microtiter plate or beads and the detecting includes
reading the fluorescence with a fluorometer.
62. The method of claim 61, wherein the detecting further comprises
detecting the fluorescence using a high throughput scanning.
63. The method of claim 55, wherein the monomers are selected from
HLA-D, DR, DP or DQ subclasses.
64. The method of claim 47 or 55, wherein the monomers are
chimeric.
65. The method of claim 47 or 55, wherein the difference in the
binding is compared after incubating the MHC complex containing the
putative MHC binding peptide and the known peptide under conditions
comprising a dissolution-testing temperature for a time sufficient
to indicate the relative dissolution rate of the putative
MHC-binding peptide.
66. The method of claim 65, wherein the dissolution-testing
temperature is in the range from about 4.degree. C. to about
37.degree. C.
67. The method of claim 65, wherein the time is from about 2 hours
to about 48 hours.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
10/269,473, filed Oct. 11, 2002, the contents of which is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to the field of
immunoassays, especially using immunoassays to detect and measure
binding of peptides to MHC alleles.
BACKGROUND OF THE INVENTION
[0003] The Class I histocompatibility ternary complex consists of
three parts associated by noncovalent bonds. A transmembrane
protein, called the MHC heavy chain is mostly exposed at the cell
surface. The cell surface domains of the MHC heavy chain contain
two segments of alpha helix that form two ridges with a groove
between them. A short peptide binds noncovalently ("fits") into
this groove between the two alpha helices, and a molecule of beta-2
microglobulin binds noncovalently along side the outer two domains
of the MHC monomer, forming a ternary complex. Peptides that bind
noncovalently to one MHC subtype heavy chain usually will not bind
to another subtype. However, all bind with the same type of beta-2
microglobulin. MHC molecules are synthesized and displayed by most
of the cells of the body.
[0004] In humans, MHC molecules are referred to as HLA molecules.
Humans primarily synthesize three different sub-types of MHC class
I molecules designated HLA-A HLA-B and HLA-C, differing only in the
heavy chains.
[0005] The MHC works coordinately with a specialized type of T cell
(the cytotoxic T cell) to rid the body of "nonself" or foreign
viral proteins. The antigen receptor on T-cells recognizes an
epitope that is a mosaic of the bound peptide and portions of the
alpha helices that make up the groove flanking it. Following
generation of peptide fragments by cleavage of a foreign protein,
the presentation of peptide fragments by the MHC molecule allows
for MHC-restricted cytotoxic T cells to survey cells for the
expression of "nonself" or foreign viral proteins. A functional
T-cell will exhibit a cytotoxic immune response upon recognition of
an MHC molecule containing bound antigenic peptide for which the
T-cell is specific.
[0006] In the performance of these functions in humans, HLA-A, B,
and C heavy chains interact with a multitude of peptides of about 8
to about 10, possibly about 8 to about 11, or about 8 to about 12
amino acids in length. Only certain peptides bind into the binding
pocket in the heavy chain of each HLA class I sub-type as the
monomer folds, although certain subtypes cross-react. By 1995,
complete coding region sequences had been determined for each of 43
HLA-A, 89 HLA-B and 11 HLA-C alleles (P. Parham et al., Immunology
Review 143:141-180, 1995).
[0007] Class II histocompatibility molecules consist of two
transmembrane polypeptides that interact to form a groove at their
outer end which, like the groove in class I molecules,
non-covalently associates with an antigenic peptide. However, the
antigenic peptides bound to class II molecules are derived from
antigens that the cell has taken in from its surroundings. In
addition, peptides that bind to class II histocompatibility
molecules are about 10 to about 30 amino acids, for example about
12 to about 24 amino acids in length (Marsh, S. G. E. et al. (2000)
The HLA Facts Book, Academic Press, p. 58-59).
[0008] Only cells, such as macrophages, dendritic cells and
B-lymphocytes, that specialize in taking up antigen from
extracellular fluids, express class II molecules.
[0009] It has long been thought that discovery of which antigen
fragments will be recognized by class I MHC-restricted T-cells can
lead to development of effective vaccines against cancer and viral
infections. A number of approaches have been developed wherein
algorithms are used to predict the amino acid sequence of HLA A, B,
or C-binding peptides and several are available on the Internet.
For example, U.S. Pat. No. 6,037,135 describes a matrix-based
algorithm that ranks peptides for likelihood of binding to any
given HLA-A allele. Similarly, most prediction methods are limited
to a set of alleles. Consequently, the predicted peptides may not
bind to MHC monomers from a whole population of patients and thus
may not be globally effective. Algorithms for HLA-class II
molecules have also been described, such as EPIMATRIX, SYFPEITHI,
TEPITOPE, PROPRED, and EPIPREDICT.
[0010] Another approach to identifying MHC-binding peptides uses a
competition-based binding assay. All competition assays yield a
comparison of binding affinities of different peptides. However,
such assays do not yield an absolute dissociation constant since
the result is dependent on the reference peptide used.
[0011] Still another approach used for determining MHC-binding
peptides is the classical reconstitution assay, e.g. using "T2"
cells, in which cells expressing an appropriate MHC allele are
"stripped" of a native binding peptide by incubating at pH 2-3 for
a short period of time for a Class I molecule and pH 4.5-5.5 for a
class II molecule. Then, to determine the binding affinity of a
putative MHC-binding peptide for the same MHC allele, the stripped
MHC monomer is combined in solution with the putative MHC-binding
peptide, and beta2-microglobulin in the case of a class I monomer,
and a conformation-dependent monoclonal antibody. The difference in
fluorescence intensity determined between cells incubated with and
without the test binding peptide after labeling, for example,
either directly with the labeled monoclonal antibody or a
fluorescence-labeled secondary antibody, can be used to determine
binding of the test peptide. However, soluble MHC monomers stripped
at low pH aggregate immediately, making their use in high
through-put assays difficult and impractical.
[0012] There are currently a series of in vitro assays for
cell-mediated immunity that use cells from the donor. The assays
include situations where the cells are from the donor, however,
many assays provide a source of antigen presenting cells from other
sources, e.g., B cell lines. These in vitro assays include the
cytotoxic T lymphocyte assay; lymphoproliferative assays, e.g.,
tritiated thymidine incorporation; the protein kinase assays, the
ion transport assay and the lymphocyte migration inhibition
function assay (Hickling, J. K. et al, J. Virol., 61: 3463 (1987);
Hengel, H. et al, J. Immunol., 139: 4196 (1987); Thorley-Lawson, D.
A. et al, Proc. Natl. Acad. Sci. USA, 84: 5384 (1987); Kadival, G.
J. et al, J. Immunol., 139: 2447 (1987); Samuelson, L. E. et al, J.
Immunol., 139: 2708 (1987); Cason, J. et al, J. Immunol. Meth.,
102: 109 (1987); and Tsein, R. J. et al, Nature, 293: 68 (1982)).
These assays are disadvantageous in that they may lack true
specificity for cell mediated immunity activity, they require
antigen processing and presentation by an APC of the same MHC type,
they are slow (sometimes lasting several days), and some are
subjective and/or require the use of radioisotopes.
[0013] Yet another approach to identifying MHC class I or class
II-binding peptides utilizes formation of MHC tetramers, which are
complexes of four MHC monomers with streptavidin, a molecule having
tetrameric binding sites for biotin, to which is bound a
fluorochrome, e.g., phycoerythrin. For example, for class I
monomers, soluble subunits of .beta.2-microglobulin, the peptide
fragment containing a putative T-cell epitope, and of a MHC heavy
chain or MHC corresponding to the predicted MHC subtype of the
peptide fragment of interest, are obtained by expression of the
polypeptides in host cells. Each of the four monomers contained in
the MHC tetramer is produced as a monomer, e.g., by refolding these
soluble subunits under conditions that favor assembly of the
soluble units into reconstituted monomers, each containing a
beta2-microglobulin, a peptide fragment, and the corresponding MHC
heavy chain. An MHC tetramer is constructed from the monomers by
biotinylation of the monomers and subsequent contact of the
biotinylated reconstituted monomers with fluorochrome-labeled
streptavidin. When contacted with a diverse population of T cells,
such as is contained in a sample of the peripheral blood
lymphocytes (PBLs) of a subject, those tetramers containing
reconstituted monomers that are recognized by a T cell in the
sample will bind to the matched T cell. Contents of the reaction is
analyzed using fluorescence flow cytometry, to determine, quantify
and/or isolate those T-cells having a MHC tetramer bound thereto
(See U.S. Pat. No. 5,635,363).
[0014] At least one other test is required to determine whether a
test peptide recognized by a T-cell by the MHC tetramer assay will
activate the T-cell to generate an immune response, a so-called
"functional test". The enzyme-linked immunospot (ELISpot) assay has
been adapted for the detection of individual cells secreting
specific cytokines or other effector molecules by attachment of a
monoclonal antibody specific for a cytokine or effector molecule on
a microplate. Cells stimulated by an antigen are contacted with the
immobilized antibody. After washing away cells and any unbound
substances, a tagged polyclonal antibody or more often, a
monoclonal antibody, specific for the same cytokine or other
effector molecule is added to the wells. Following a wash, a
colorant that binds to the tagged antibody is added such that a
blue-black colored precipitate (or spot) forms at the sites of
cytokine localization. The spots can be counted manually or with
automated ELISpot reader system to quantitate the response. A final
confirmation of T-cell activation by the test peptide may require
in vivo testing, for example in a mouse model. Thus, the route to
final confirmation of the efficacy of a MHC-binding peptide is
expensive and time consuming.
[0015] A similar procedure is followed for formation of MHC class
II tetramers, except that alpha and beta chains are not produced by
E coli but are produced on insect cells. The insect cells are
transfected with alpha and beta chains to be secreted in the
supernatant. The secreted molecules are empty and can be loaded
with the desired MHC-binding peptide. After peptide loading the
monomer is biotinylated and tetramerized with the streptavidin
conjugated to a fluorochrome.
[0016] Thus, there is still a need in the art for new and better
systems and methods for preliminary screening assays identifying
putative MHC class I and class II-binding peptides and for
measuring peptide binding to MHC alleles, such as HLA-A, B, C, or
D, DR, DP or DQ, especially an in vitro assay in solid phase
format. There is also a need in the art to develop methods to
determine the MHC-binding affinity of MHC-binding peptides and for
a measurement for the dissociation rate of a bound peptide from the
MHC molecule.
SUMMARY OF THE INVENTION
[0017] The present invention is based on the discovery that MHC
class I and class II monomers when immobilized to a solid surface
are still capable of incorporating from solution an MHC-binding
peptide and forming an MHC complex.
[0018] Accordingly, in one embodiment the invention provides a
system comprising a solid surface, wherein the surface has attached
thereto one or more MHC class I or class II monomer or modified MHC
class I or class II monomer. The class I monomers denature in a
denaturing condition and reconstitutes to form a ternary complex
containing a suitable MHC-binding peptide in the binding pocket
under reconstituting conditions. The MHC class II monomers bind an
MHC-binding peptide from solution within the pH range from 4 to
about 8. In another embodiment kits comprising the invention
systems are also provided.
[0019] In another embodiment, the invention provides methods for
determining binding between a MHC class I monomer or modified MHC
class I monomer and a putative MHC-binding peptide that include
incubating a solid surface having attached thereto a plurality of
previously denatured MHC class I monomers or modified MHC class I
monomers under reconstituting conditions in the presence and
absence of the putative MHC-binding peptide such that the monomers
reconstitute to form a ternary complex containing a suitable
MHC-binding peptide under the reconstituting conditions. Binding to
the ternary complex of a monoclonal antibody that does not bind to
dissociated components of the MHC complex indicates binding between
the putative MHC-binding peptide and the monomers. Another approach
comprises use of a fluorescently labeled peptide to determine
whether the unknown peptide can compete with the fluorescently
labeled peptide for binding to the HLA molecule.
[0020] In still another embodiment, the invention provides methods
for determining binding between a MHC class II monomer or modified
MHC class II monomer and a putative MHC-binding peptide therefor by
incubating under suitable peptide loading conditions a solid
surface having attached thereto a plurality of the MHC monomers or
modified MHC monomers in the presence and absence of the putative
MHC-binding peptide to form a complex containing a suitable
MHC-binding peptide, and determining binding of the MHC monomers
with the putative MHC-binding peptide.
[0021] In yet another embodiment, the invention provides methods
for determining the degree of binding affinity of an MHC class I
monomer or modified MHC class I monomer for a putative MHC-binding
peptide therefor by incubating, under reconstituting conditions, at
least one such denatured MHC monomer or modified MHC monomer
attached to a solid surface with the putative MHC-binding peptide
and a monoclonal antibody that specifically binds to a
conformational epitope in a first complex containing a
corresponding reconstituted MHC monomer and does not bind to any
dissociated component of the MHC complex, and comparing binding of
the monoclonal antibody to the MHC complex that contains the
putative MHC-binding peptide with binding of the monoclonal
antibody to a corresponding complex containing the monomer and a
known MHC-binding peptide. A difference in the bindings indicates
the relative degree of binding affinity of the reconstituted
monomer for the putative MHC-binding peptide.
[0022] In still another embodiment, the invention provides methods
for determining the degree of binding affinity of an MHC class II
monomer or modified MHC class II monomer for a putative MHC-binding
peptide therefor by incubating under suitable peptide loading
conditions at least one MHC class II monomer or modified MHC class
II monomer attached to a solid surface with the putative
MHC-binding peptide and a monoclonal antibody that specifically
binds to an epitope in a first complex containing the monomer and
does not bind to any dissociated component of the MHC complex; and
comparing binding of the monoclonal antibody to the MHC complex
that contains the putative MHC-binding peptide with binding of the
monoclonal antibody to a corresponding complex containing the
monomer and a known MHC-binding peptide. A difference in the
bindings indicates the relative degree of binding affinity of the
monomer for the putative MHC-binding peptide.
[0023] In still another embodiment, the invention provides methods
for determining the stability at different temperatures, but in
particular at 37.degree. C., of an MHC monomer or modified MHC
monomer for a putative MHC-binding peptide therefor. In this
embodiment, at least one denatured MHC monomer or modified MHC
monomer attached to a solid surface is incubated under
reconstituting conditions with the putative MHC-binding peptide and
a monoclonal antibody that specifically binds to a conformational
epitope of a corresponding reconstituted MHC monomer that is not
present in the denatured monomer. After the reconstituted ternary
complex with the monoclonal antibody is incubated at different
temperatures and different times. The difference in the signal
obtained at different temperatures and different times, indicates
the relative stability of the reconstituted monomer for the
putative MHC-binding peptide.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1 is a schematic representation of the immunoassay.
[0025] FIG. 2 is a graph showing calibration of the anti-HLA-class
I-FITC mAb for fluorometric assay.
[0026] FIG. 3 is a graph showing a decrease in binding of
anti-HLA-class I-FITC mAb to a reconstituted HLA heavy chain
monomer Mart1 26-35 with increasing temperature as determined by
fluorescence of bound antibody.
[0027] FIG. 4 is a graph showing the binding of an anti-HLA-class
I-FITC monoclonal antibody to human and mouse alleles as determined
by fluorescence of bound antibody.
[0028] FIG. 5 is a graph showing renaturation in various buffer
solutions of the MHC heavy chain monomers attached to a plate as
detected by an anti-HLA-class I-FITC mAb.
[0029] FIG. 6 is a graph showing antibody binding to monomer at
concentrations of anti-HLA-class I mAb of 1 to 2 .mu.g/ml for
various HLA heavy chain monomer concentrations to determine the
optimal concentration of the anti-HLA-class I antibody for use with
a microtiter plate assay.
[0030] FIGS. 7A and 7B are graphs showing the dose response curve
obtained with two different HLA heavy chain monomers. FIG. 7A shows
the results with HLA-A*0201/Mart1 2635L (Linear regression
equation: y=1555.5x +39.787; R2=0.9889. FIG. 7B shows the results
with HLA heavy chain monomer HLA-A*0201/HIVpol (Linear regression
equation: y=1487.1X +13.927, R2=0.9982)
[0031] FIG. 8 is a graph showing the specificity of the
anti-HLA-class I antibody for various HLA-A and HLA-B alleles.
[0032] FIG. 9 is a schematic drawing showing formation of a
human-mouse chimeric MHC modified monomer according to the
invention.
[0033] FIGS. 10A-D show graphs of the dissociation curves for
renatured peptides (HBV core peptide; 26-35L; 26-35; 27-35,
respectively).
[0034] FIGS. 10E-H show graphs of the off rates for peptides HBV
core; 26-35L; 26-35; and 27-35, respectively.
[0035] FIG. 10I shows the effect of temperature on monomer
dissociation.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention relates in general to immunoassays
directed to detection and measurement of the binding affinity of
MHC class I and class II monomers, especially MHC class I and class
II monomers immobilized on a surface, for putative MHC-binding
peptides. It is the discovery of the present invention that MHC
monomers and modified MHC monomers immobilized to a solid surface
are still capable of refolding so as to form an MHC complex by bind
from solution a MHC-binding peptide that has the requisite binding
affinity. Moreover, it is the discovery of the present invention
that such binding can be detected in an immunoassay format, such as
one utilizing a conformation-dependent monoclonal antibody that
specifically binds to an MHC complex containing such refolded or
reconstituted MHC monomers but does not bind to dissociated
components of the MHC complex.
[0037] As used herein, the terms "MHC monomer" and "HLA monomer",
when used to refer to a MHC class I molecule, mean a class I heavy
chain that maintains the ability to assemble into a ternary complex
with an appropriate MHC-binding or HLA-binding peptide and beta-2
microglobulin under renaturing conditions. These terms are also
used to refer to the denatured forms of the Class I monomers that
result from subjecting the respective complexes to denaturing
conditions, causing the monomer to unfold and dissociate from a
MHC-binding peptide (and from beta-2 microglobulin for a class I
monomer). When used to refer to a MHC class II molecule, the terms
"MHC monomer" and HLA monomer"mean alpha and beta class II chains
that maintain the ability to assemble into a heterodimer that forms
an MHC complex to bind an appropriate MHC-binding or HLA-binding
peptide from solution under suitable peptide loading conditions.
Class II monomers do not need to be denatured and renatured in the
presence of the MHC binding peptide to bind such peptides from
solution, for example at a pH in the range from about 4 to about
8.
[0038] As used herein, the terms "modified MHC monomer" and
"modified HLA monomer" refer to class I and class II monomers as
described above, but which have been engineered to introduce
modifications as described below. These terms also encompass
functional fragments of the MHC monomer that maintain the ability
to assemble into an MHC complex with an appropriate MHC-binding or
HLA-binding peptide (and beta-2 microglobulin for class I monomers)
under renaturing conditions and to dissociate under denaturing
conditions. For example, a functional fragment can comprise only
the .alpha..sub.1, .alpha..sub.2, .alpha..sub.3, domains, or only
.alpha..sub.1, .alpha..sub.2 domains, of the class I heavy chain,
i.e., the cell surface domains, that participate in formation of
the ternary complex.
[0039] MHC class II monomers are composed of two transmembrane
polypeptide chains, each containing an extracellular domain, a
transmembrane domain and an intracellular tail. The extracellular
domains bind together to form a MHC II heterodimer. Each of these
polypeptides (known as alpha and beta) folds to form two separate
extracellular domains; alpha-1 and alpha-2 for the alpha
polypeptide, and beta-1 and beta-2 for the beta polypeptide.
Between the alpha-1 and beta-1 domains lies a region known as the
binding pocket or "groove," which is bounded by a beta-pleated
sheet on the bottom and two alpha helices on the sides, and is
capable of binding (via non-covalent interactions) an MHC II
binding peptide. This MHC II binding peptide is "presented" to a
T.sub.H-cell and defines the antigen "epitope" that the
T.sub.H-cell recognizes. Modified MHC II monomers can be
heterodimers containing only the extracellular domains of the alpha
and beta chains.
[0040] In another embodiment, modified MHC monomers can be class I
heavy chain molecules, or functional fragments thereof, contained
in a fusion protein or "single chain" molecule and may further
include an amino acid sequence functioning as a linker between cell
surface domains of the monomer, a detectable marker or as a ligand
to attach the molecule to a solid support that is coated with a
second ligand with which the ligand in the fusion protein reacts.
Moreover the terms "modified MHC monomer" and "modified HLA
monomer" are intended to encompass chimera containing domains of
MHC class I and class II molecules from more than one species or
from more than one class I or class II subclass. FIG. 9 herein
illustrates preparation of a chimera by substitution of a mouse
H-2Kb domain for one of the three alpha domains in a human HLA-A2
fragment. Such a molecule is conveniently expressed as a single
chain with optional amino acid linkers between subunits or as a
fusion protein as is known in the art.
[0041] In another embodiment, modified MHC monomers can be class II
heterodimers containing only the extracellular domains of the alpha
and beta chains. Each polypeptide chain can additionally have added
to the C-terminus a leucine zipper sequence (e.g. either Fos or
Jun) and one of the chains can be further modified to add an amino
acid segment containing a natural recognition site of BirA enzyme
that is found in MHC II DR4 .beta. chain as a biotinylation tag at
the C-terminal end of the fusion proteins.
[0042] Preparation of monomers
[0043] Located on chromosome 6 in humans, the class I MHC has three
loci, HLA-, HLA-B, and HLA-C. The first two loci have a large
number of alleles encoding alloantigens. These are found to consist
of a 44 Kd heavy chain subunit and a 12 Kd beta.sub.2-microglobulin
subunit, which is common to all antigenic specificities. For
example, soluble HLA-A2 can be purified after papain digestion of
plasma membranes from the homozygous human lymphoblastoid cell line
J-Y as described by Turner, M. J. et al., J. Biol. Chem. (1977)
252:7555-7567. Papain cleaves the 44 Kd heavy chain close to the
transmembrane region, yielding a molecule comprised of
.alpha..sub.1, .alpha..sub.2, and .alpha..sub.3 domains and beta-2
microglobulin.
[0044] The MHC monomers can be isolated from appropriate cells or
can be recombinantly produced, for example as described by Paul et
al, Fundamental Immunology, 2d Ed., W. E. Paul, ed., Ravens Press
N.Y. 1989, Chapters 16-18) and readily modified, as described
below.
[0045] The term "isolated" as applied to MHC monomers herein refers
to an MHC glycoprotein of MHC class I or class II, that is in other
than its native state, for example, not associated with the cell
membrane of a cell that normally expresses MHC. This term embraces
a full-length subunit chain, as well as a functional fragment of
the MHC monomer. A functional fragment is one comprising an antigen
binding site and sequences necessary for recognition by the
appropriate T cell receptor. It typically comprises at least about
60-80%, typically 90-95% of the sequence of the full-length chain.
As described herein, the "isolated" MHC subunit component may be
recombinantly produced or solubilized from the appropriate cell
source.
[0046] It is well known that native forms of "mature" MHC
glycoprotein monomers will vary somewhat in length because of
deletions, substitutions, and insertions or additions of one or
more amino acids in the sequences. Thus, MHC monomers are subject
to substantial natural modification, yet are still capable of
retaining their functions. Modified protein chains can also be
readily designed and manufactured utilizing various recombinant DNA
techniques well known to those skilled in the art and described in
detail, below. For example, the chains can vary from the naturally
occurring sequence at the primary structure level by amino acid
substitutions, additions, deletions, and the like. These
modifications can be used in a number of combinations to produce
the final modified protein chain.
[0047] In general, modifications of the genes encoding the MHC
monomer may be readily accomplished by a variety of well-known
techniques, such as site-directed mutagenesis. The effect of any
particular modification can be evaluated by routine screening in a
suitable assay for the desired characteristic. For instance, a
change in the immunological character of the subunit can be
detected by competitive immunoassay with an appropriate antibody.
The effect of a modification on the ability of the monomer to
activate T cells can be tested using standard in vitro cellular
assays or the methods described in the example section, below.
Modifications of other properties such as redox or thermal
stability, hydrophobicity, susceptibility to proteolysis, or the
tendency to aggregate are all assayed according to standard
techniques.
[0048] This invention provides amino acid sequence modification of
MHC monomers prepared with various objectives in mind, including
increasing the affinity of the subunit for antigenic peptides
and/or T cell receptors, facilitating the stability, purification
and preparation of the subunits. The monomers may also be modified
to modify plasma half-life, improve therapeutic efficacy, or to
lessen the severity or occurrence of side effects during
therapeutic use of complexes of the present invention. The amino
acid sequence modifications of the subunits are usually
predetermined variants not found in nature or naturally occurring
alleles. The variants typically exhibit the same biological
activity (for example, MHC-peptide binding) as the naturally
occurring analogue.
[0049] Insertional modifications of the present invention are those
in which one or more amino acid residues are introduced into a
predetermined site in the MHC monomer and which displace the
preexisting residues. For instance, insertional modifications can
be fusions of heterologous proteins or polypeptides to the amino or
carboxyl terminus of the subunits.
[0050] Other modifications include fusions of the monomer with a
heterologous signal sequence and fusions of the monomer to
polypeptides having enhanced plasma half-life (ordinarily>about
20 hours) such as immunoglobulin chains or fragments thereof as is
known in the art.
[0051] Substitutional modifications are those in which at least one
residue has been removed and a different residue inserted in its
place. Nonnatural amino acid (i.e., amino acids not normally found
in native proteins), as well as isosteric analogs (amino acid or
otherwise) are also suitable for use in this invention.
[0052] Substantial changes in function or immunological identity
are made by selecting substituting residues that differ in their
effect on maintaining the structure of the polypeptide backbone
(e.g., as a sheet or helical conformation), the charge or
hydrophobicity of the molecule at the target site, or the bulk of
the side chain. The substitutions which in general are expected to
produce the greatest changes in function will be those in which (a)
a hydrophilic residue, e.g., serine or threonine, is substituted
for (or by) a hydrophobic residue, e.g. leucine, isoleucine,
phenylalanine, valine or alanine; (b) a cysteine or proline is
substituted for (or by) any other residue; (c) a residue having an
electropositive side chain, e.g., lysine, arginine, or histidine,
is substituted for (or by) an electronegative residue, e.g.,
glutamine or aspartine; or (d) a residue having a bulky side chain,
e.g., phenylalanine, is substituted for (or by) one not having a
side chain, e.g., glycine.
[0053] Substitutional modifications of the monomers also include
those where functionally homologous (having at least about 70%
homology) domains of other proteins are substituted by routine
methods for one or more of the MHC subunit domains. Particularly
preferred proteins for this purpose are domains from other species,
such as murine species as illustrated in FIG. 9 herein.
[0054] Another class of modifications is deletional modifications.
Deletions are characterized by the removal of one or more amino
acid residues from the MHC monomer sequence. Typically, the
transmembrane and cytoplasmic domains are deleted. Deletions of
cysteine or other labile residues also may be desirable, for
example in increasing the oxidative stability of the MHC complex.
Deletion or substitutions of potential proteolysis sites, e.g.,
ArgArg, is accomplished by deleting one of the basic residues or
substituting one by glutaminyl or histidyl residues.
[0055] A preferred class of substitutional or deletional
modifications comprises those involving the transmembrane region of
the subunit. Transmembrane regions of MHC monomers are highly
hydrophobic or lipophilic domains that are the proper size to span
the lipid bilayer of the cellular membrane. They are believed to
anchor the MHC molecule in the cell membrane. Inactivation of the
transmembrane domain, typically by deletion or substitution of
transmembrane domain hydroxylation residues, will facilitate
recovery and formulation by reducing its cellular or membrane lipid
affinity and improving its aqueous solubility. Alternatively, the
transmembrane and cytoplasmic domains can be deleted to avoid the
introduction of potentially immunogenic epitopes. Inactivation of
the membrane binding function is accomplished by deletion of
sufficient residues to produce a substantially hydrophilic
hydropathy profile at this site or by substitution with
heterologous residues, which accomplish the same result.
[0056] A principal advantage of the transmembrane-inactivated MHC
monomer is that it may be secreted into the culture medium of
recombinant hosts. This variant is soluble in body fluids such as
blood and does not have an appreciable affinity for cell membrane
lipids, thus considerably simplifying its recovery from recombinant
cell culture. Typically, modified MHC monomers of this invention
will not have a functional transmembrane domain and preferably will
not have a functional cytoplasmic sequence. Such modified MHC
monomers will consist essentially of the effective portion of the
extracellular domain of the MHC monomer. In some circumstances, the
monomer comprises sequences from the transmembrane region (up to
about 10 amino acids), so long as solubility is not significantly
affected.
[0057] For example, the transmembrane domain may be substituted by
any amino acid sequence, e.g., a random or predetermined sequence
of about 5 to 50 serine, threonine, lysine, arginine, glutamine,
aspartic acid and like hydrophilic residues, which altogether
exhibit a hydrophilic hydropathy profile. Like the deletional
(truncated) monomer, these monomers are secreted into the culture
medium of recombinant hosts.
[0058] Glycosylation variants are included within the scope of this
invention. They include variants completely lacking in
glycosylation (unglycosylated) and variants having at least one
less glycosylated site than the native form (deglycosylated) as
well as variants in which the glycosylation has been changed.
Included are deglycosylated and unglycosylated amino acid sequence
variants, deglycosylated and unglycosylated subunits having the
native, unmodified amino acid sequence. For example, substitutional
or deletional mutagenesis is employed to eliminate the N- or
O-linked glycosylation sites of the subunit, e.g., the asparagine
residue is deleted or substituted for by another basic residue such
as lysine or histidine. Alternatively, flanking residues making up
the glycosylation site are substituted or deleted, even though the
asparagine residues remain unchanged, in order to prevent
glycosylation by eliminating the glycosylation recognition site.
Additionally, unglycosylated MHC monomers that have the amino acid
sequence of the native monomers are produced in recombinant
prokaryotic cell culture because prokaryotes are incapable of
introducing glycosylation into polypeptides.
[0059] Glycosylation variants are conveniently produced by
selecting appropriate host cells or by in vitro methods. Yeast, for
example, introduce glycosylation which varies significantly from
that of mammalian systems. Similarly, mammalian cells having a
different species (e.g., hamster, murine, insect, porcine, bovine
or ovine) or tissue origin (e.g., lung, liver, lymphoid,
mesenchymal or epidermal) than the MHC source are routinely
screened for the ability to introduce variant glycosylation as
characterized for example by elevated levels of mannose or variant
ratios of mannose, fucose, sialic acid, and other sugars typically
found in mammalian glycoproteins. In vitro processing of the
subunit typically is accomplished by enzymatic hydrolysis, e.g.,
neuraminidase digestion.
[0060] MHC glycoproteins suitable for use in the present invention
have been isolated from a multiplicity of cells using a variety of
techniques including solubilization by treatment with papain, by
treatment with 3M KCl, and by treatment with detergent. For
example, detergent extraction of class I protein followed by
affinity purification can be used. Detergent can then be removed by
dialysis or selective binding beads. The molecules can be obtained
by isolation from any MHC I bearing cell, for example from an
individual suffering from a targeted cancer or viral disease.
[0061] Isolation of individual heavy chain from the isolated MHC
glycoproteins is easily achieved using standard techniques known to
those skilled in the art. For example, the heavy chain can be
separated using SDS/PAGE and electroelution of the heavy chain from
the gel (see, e.g., Dornmair et al., supra and Hunkapiller, et al.,
Methods in Enzymol. 91:227-236 (1983). Separate subunits from MHC
molecules are also isolated using SDS/PAGE followed by
electroelution as described in Gorga et al. J. Biol. Chem.
262:16087-16094 (1987) and Dornmair et al. Cold Spring Harbor Symp.
Quant. Biol. 54:409-416 (1989). Those of skill will recognize that
a number of other standard methods of separating molecules can be
used, such as ion exchange chromatography, size exclusion
chromatography or affinity chromatography.
[0062] Alternatively, the amino acid sequences of a number of MHC
monomer proteins are known, and the genes have been cloned;
therefore, the monomers can be expressed using recombinant methods.
These techniques allow a number of modifications of the MHC
monomers as described above. For instance, recombinant techniques
provide methods for carboxy terminal truncation, which deletes the
hydrophobic transmembrane domain. The carboxy termini can also be
arbitrarily chosen to facilitate the conjugation of ligands or
labels, for example, by introducing cysteine and/or lysine residues
into the molecule. The synthetic gene will typically include
restriction sites to aid insertion into expression vectors and
manipulation of the gene sequence. The genes encoding the
appropriate monomers are then inserted into expression vectors,
expressed in an appropriate host, such as E. coli, yeast, insect,
or other suitable cells, and the recombinant proteins are
obtained.
[0063] As the availability of the gene permits ready manipulation
of the sequence, a second generation of construction includes
chimeric constructs, for example as illustrated in FIG. 9. The
.alpha..sub.1, .alpha..sub.2, .alpha..sub.3, domains of the class I
heavy chain are linked typically by the .alpha..sub.3 domain of
class I with beta-2 microglobulin and coexpressed to stabilize the
MHC complex. The transmembrane and intracellular domains of the
class I gene can optionally also be included.
[0064] Construction of expression vectors and recombinant
production from the appropriate DNA sequences are performed by
methods known in the art. Standard techniques are used for DNA and
RNA isolation, amplification, and cloning. Generally enzymatic
reactions involving DNA ligase, DNA polymerase, restriction
endonucleases, and the like, are performed according to the
manufacturer's specifications. These techniques and various other
techniques are generally performed according to Sambrook et al.,
Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989. The procedures therein
are believed to be well known in the art.
[0065] Expression can be in procaryotic or eucaryotic systems.
Suitable eucaryotic systems include yeast, plant and insect
systems, such as the Drosophila expression vectors under an
inducible promoter. Procaryotes most frequently are represented by
various strains of E. coli. However, other microbial strains may
also be used, such as bacilli, for example Bacillus subtilis,
various species of Pseudomonas, or other bacterial strains. In such
procaryotic systems, plasmid vectors that contain replication sites
and control sequences derived from a species compatible with the
host are used. For example, E. coli is typically transformed using
derivatives of pBR322, a plasmid derived from an E. coli species by
Bolivar et al., Gene (1977) 2:95. Commonly used procaryotic control
sequences, which are defined herein to include promoters for
transcription initiation, optionally with an operator, along with
ribosome binding site sequences, including such commonly used
promoters as the .beta.-lactamase (penicillinase) and lactose (lac)
promoter systems (Change et al., Nature (1977) 198:1056) and the
tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids
Res. (1980) 8:4057) and the lambda-derived P.sub.L promoter and
N-gene ribosome binding site (Shimatake et al., Nature (1981)
292:128). Any available promoter system compatible with procaryotes
can be used.
[0066] The expression systems useful in the eucaryotic hosts
comprise promoters derived from appropriate eucaryotic genes. A
class of promoters useful in yeast, for example, includes promoters
for synthesis of glycolytic enzymes, including those for
3-phosphoglycerate kinase (Hitzeman, et al., J. Biol. Chem. (1980)
255:2073). Other promoters include, for example, those from the
enolase gene (Holland, M. J., et al. J. Biol. Chem. (1981)
256:1385) or the Leu2 gene obtained from YEp13 (Broach, J., et al.,
Gene (1978) 8:121). A Drosophila expression system under an
inducible promoter (Invitrogen, San Diego, Calif.) can also be
used.
[0067] Suitable mammalian promoters include the early and late
promoters from SV40 (Fiers, et al., Nature (1978) 273:113) or other
viral promoters such as those derived from polyoma, adenovirus II,
bovine papilloma virus or avian sarcoma viruses. Suitable viral and
mammalian enhancers are cited above.
[0068] The expression system is constructed from the foregoing
control elements operably linked to the MHC sequences using
standard methods, employing standard ligation and restriction
techniques, which are well understood in the art. Isolated
plasmids, DNA sequences, or synthesized oligonucleotides are
cleaved, tailored, and religated in the form desired.
[0069] Site-specific DNA cleavage is performed by treatment with
the suitable restriction enzyme (or enzymes) under conditions which
are generally understood in the art, and the particulars of which
are specified by the manufacturer of these commercially available
restriction enzymes. In general, about 1 .mu.g of plasmid or DNA
sequence is cleaved by one unit of enzyme in about 20 .mu.l of
buffer solution; an excess of restriction enzyme may be used to
insure complete digestion of the DNA substrate. After each
incubation, protein is removed by extraction with
phenol/chloroform, and may be followed by ether extraction, and the
nucleic acid recovered from aqueous fractions by precipitation with
ethanol followed by running over a Sephadex G-50 spin column. If
desired, size separation of the cleaved fragments may be
performed.
[0070] Restriction cleaved fragments may be blunt ended by treating
with the large fragment of E. coli DNA polymerase I (Klenow) in the
presence of the four deoxynucleotide triphosphates (dNTPs). After
treatment with Klenow, the mixture is extracted with
phenol/chloroform and ethanol precipitated followed by running over
a Sephadex G-50 spin column.
[0071] Synthetic oligonucleotides are prepared using commercially
available automated oligonucleotide synthesizers. In the proteins
of the invention, however, a synthetic gene is conveniently
employed. The gene design can include restriction sites that permit
easy manipulation of the gene to replace coding sequence portions
with these encoding analogs.
[0072] Correct ligations for plasmid construction can be confirmed
by first transforming E. coli strain MM294 obtained from E. coli
Genetic Stock Center, CGSC #6135, or other suitable host, with the
ligation mixture. Successful transformants can be selected by
ampicillin, tetracycline or other antibiotic resistance or by using
other markers depending on the mode of plasmid construction, as is
understood in the art. Plasmids from the transformants are then
prepared, optionally following chloramphenicol amplification. The
isolated DNA is analyzed by restriction and/or sequenced by the
dideoxy method of Sanger, F., et al., Proc. Natl. Acad. Sci. USA
(1977) 74:5463 as further described by Messing, et al., Nucleic
Acids Res. (1981) 9:309, or by the method of Maxam, et al., Methods
in Enzymology (1980) 65:499.
[0073] The constructed vector is then transformed into a suitable
host for production of the protein. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride, as
described by Cohen, S. N., Proc. Natl. Acad. Sci. USA (1972)
69:2110, or the RbCl method described in Maniatis, et al.,
Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor
Press, p. 254 is used for procaryotes or other cells which contain
substantial cell wall barriers. For mammalian cells without such
cell walls, the calcium phosphate precipitation method of Graham
and van der Eb, Virology (1978) 52:546 or electroporation is
preferred. Transformations into yeast are carried out according to
the method of Van Solingen, P., et al., J. Bacter. (1977) 130:946
and Hsiao, C. L., et al., Proc. Natl. Acad. Sci. USA (1979)
76:3829.
[0074] The transformed cells are then cultured under conditions
favoring expression of the MHC sequence and the recombinantly
produced protein recovered from the culture.
[0075] MHC-binding Peptides
[0076] It is believed that the presentation of antigen by the MHC
glycoprotein on the surface of antigen-presenting cells (APCs)
occurs subsequent to the proteolysis of antigenic proteins into
smaller peptide units. The location of these smaller segments
within the antigenic protein can be determined empirically. class I
MHC-binding peptides are thought to be about 8 to about 10,
possibly about 8 to about 11, or about 8 to about 12 residues in
length. class II MHC-binding peptides are thought to be about 10 to
about 30 amino acids, for example, about 12 or to about 24 amino
acids in length. These peptides contain both the agretope
(recognized by the MHC molecule) and the epitope (recognized by T
cell receptor on the T cell). The epitope is a contiguous or
noncontiguous sequence of about 5-6 amino acids that is recognized
by the antigen-specific T cell receptor. The agretope is a
continuous or noncontiguous sequence that is responsible for
binding of the peptide with the MHC class I glycoproteins. The
invention provides systems, kits, and assays for evaluating
putative MHC-binding peptides to determine whether such fragments
can be incorporated into a ternary complex with an MHC monomer or
modified MHC monomer.
[0077] Thus, the invention provides systems, kits and screening
methods to be used in screening of candidate peptides for use in
diagnostic assays, vaccines, and other treatment modalities.
Putative MHC-binding peptides for use in the invention methods can
be made using any method known in the art, the most convenient
being peptide synthesis for fragments of 8 to 12 amino acids in
length.
[0078] Accordingly, in one embodiment the invention provides a
system comprising a solid surface having attached thereto one or
more MHC monomer or modified MHC monomer wherein the monomer
denatures in a denaturing condition and reconstitutes to form a
functional binding pocket containing a suitable MHC-binding peptide
under reconstituting conditions. For example, a plurality of the
monomers can be bound to a single surface. The surface of the
system can be any known or later discovered solid surface
including, without any limitation, any solid, polymer, membrane,
synthetic surface, and the like. For example, the solid surface of
the invention system can be a microtiter plate, such as the wells
of a microtiter plate, or a bead, such as an agarose A bead, an
agarose G bead, and the like. In one aspect, the solid surface of
the invention system is suitable for use in a high throughput
scanning system, e.g., the surface is compatible with the high
throughput system or allows a system to work with the entities
associated with the surface in a high throughput manner, such as
fluorescence determined flow cytometry.
[0079] Recently, a short peptide sequence (StrepTagII.TM.) has been
identified that demonstrates binding affinity
(Kd.about.1.times.10-6M) for the biotin-binding site of a mutated
streptavidin molecule, called StrepTactin. The molecule d-biotin,
which binds with higher affinity to strepTactin
(Kd.about.1.times.10-13 M), effectively competes with the
StrepTagII for the binding site. (Knabel, M., Franz, T. J.,
Schiemann, M., Wulf, A., Villmow, B., Schmidt, B., Bernhard, H.,
Wagner, H., Busch, D. H. (2002) Reversible MHC multimer staining
for functional isolation of T-cell populations and effective
adoptive transfer. Nature Medicine Vol. 8 No. 6, June 2002. pp:
631-637). Attachment of the MHC monomers to the solid surface can
be accomplished by any method known in the art. For example, the
solid surface can be coated with a first binding ligand, such as
avidin, and the monomer is then provided with a second binding
ligand, such as biotin, wherein the first ligand binds specifically
with the second ligand. The second binding ligand may optionally be
attached to the monomers via a C-terminal end. Attachment of the
one or more monomers to the solid surface is optionally reversible
or cleavable. For example, a cleavable binding complex is
commercially available from Amersham Bioscience Bioscience (Orsay
France) such as Factor Xa, PreScission Protease and thrombin. All
of these proteases can be used with the GST affinity tag from
proteins expressed using pGEX-T vectors.
[0080] The invention system comprising a solid support with
attached MHC class I or class II monomers. Solid supports with
attached MHC class I monomers are preferably stored in a renatured
state, by causing formation of an MHC complex with the MHC class I
monomer containing a MHC binding peptide in the binding pocket, as
described herein. For MHC class I monomers, the MHC complex is a
ternary complex additionally containing a beta-2 microglobulin
molecule bound thereto.
[0081] Formation of the MHC complex containing an MHC class I
monomer or modified MHC class I monomer attached to a solid support
is referred to herein as "renaturation" and is accomplished under
renaturing conditions as is know in the art and described herein.
For example, renaturing conditions typically include the presence
of a suitable MHC binding peptide for the monomer and a suitable
refolding buffer having a pH of from about 7 to about 8.5 for class
I MHC monomers. For class I MHC monomers, suitable renaturing
conditions also include the presence of beta-2 microglobulin.
Suitable refolding buffers are illustrated in the Examples herein
and are known in the art. In further preparation for storage, the
solid support with bound MHC monomer(s) can be dried (while in a
renatured state for class I monomers), for example by exposure to a
buffer containing sugars. In preparation for use of the solid
support of the invention system to test putative MHC-binding
peptides, the solid support and attached MHC class I monomers in
complex are exposed to denaturing conditions to cause dissociation
and unfolding of the monomers. For example, denaturing conditions
can comprise exposure of the solid support and bound monomers to a
pH of about 2 to about 4 for sufficient time to cause dissociation
of the monomer complexes without damage to the monomers.
[0082] Unlike with MHC class I monomers, peptide loading for class
II monomers takes place in the pH range from about 5 to about 8 and
can be accomplished from solution without having to denature and
renature the monomer in the presence of the peptide to be
loaded.
[0083] Optionally, the invention system may further comprise a
monoclonal antibody, described in greater detail below, that binds
specifically to a conformational epitope that is present in the MHC
complex and absent in the dissociated components of such an MHC
complex. For example, the conformational epitope may be formed in
the reconstituted MHC monomers or modified MHC monomers used in the
system and absent in the denatured monomers. Alternatively the
monoclonal antibody may differentiate between monomers that have
bound to a MHC-binding peptide and those that have not. The
invention system may further contain a supply of beta-2
microglobulin.
[0084] The MHC monomer used in the invention systems and methods
can be any MHC monomer or modified MHC monomer, e.g., class I heavy
chain, capable of binding an MHC-binding peptide, as described
herein. The MHC monomer can be encoded by any partial or
full-length modified or unmodified MHC gene sequence from any
species or subtype, or a combination thereof, including without
limitation human and murine species, and chimera thereof. Preferred
MHC encoding gene sequences are those encoding any HLA allele
genotype and any variation or polymorphism thereof. For example,
the MHC monomer utilized in the invention systems and methods can
be any partial or full-length HLA monomer that binds an HLA-binding
peptide under renaturing conditions, i.e., any subtype or allele of
HLA-A, HLA-B, HLA-C, or HLA-D.
[0085] For example, in one embodiment, the MHC monomer is modified
by truncation to include only the .alpha..sub.1, .alpha..sub.2 and
the .alpha.3 domains of an HLA class I heavy chain or the
.alpha..sub.1, .alpha..sub.2 and the .beta..sub.1, .beta..sub.2
domains of an HLA class II monomer, which bind together to form a
MHC II heterodimer. In still another embodiment, the MHC monomer
can be a chimeric, such as a fusion protein, containing these MHC
domains and an anchor domain, wherein the MHC domain binds to a
MHC-binding a peptide, as described herein, while the anchor domain
is suitable for immobilizing the MHC monomer to a surface. The
anchor domain can be a polypeptide fused with the HLA domain to
form a fusion protein or can be any entity coupled to the HLA
domain through any suitable means known in the art, e.g., a
biotinylated MHC monomer.
[0086] The MHC monomer can be attached to the solid surface by any
suitable means known in the art. For example, the MHC monomer can
be immobilized to a surface either directly or indirectly, e.g.,
via an anchoring or connecting entity. In one embodiment, the solid
surface of the invention system is coated with a first ligand
entity, which binds to or interacts with a second ligand connected
to or within the MHC monomer, e.g., via covalent or noncovalent
bond. In another embodiment, the surface is coated with avidin or
its derivatives, e.g., streptavidin, and the MHC monomer contains
biotin or its derivatives as its anchor domain. Attachment of the
MHC monomer to the solid surface, in one embodiment of the
invention, is reversible or cleavable.
[0087] The MHC monomer coated or immobilized to a solid surface can
be denatured, e.g., stripped or dissociated in a denaturing
condition, and then renatured, e.g., refolded from a denatured form
under a non-denaturing or renaturing condition so as to bind an
appropriate MHC-binding peptide. In one embodiment, the surface
coated with the MHC monomer provided by the present invention can
be dried and stored for use at a later time. Preferably, the
storage is at about 4 degrees C.
[0088] In addition to the surface coated with the MHC monomer, the
system of the invention can further include a monoclonal antibody
and a peptide. The peptide can be any peptide that binds to the HLA
monomers, e.g., MHC-binding peptides. In one embodiment, the
peptide has high affinity to the MHC monomer, e.g., HBc high
affinity peptide.
[0089] The monoclonal antibody used in the invention systems and
methods can be any monoclonal antibody that specifically binds to
an epitope present only in a complex of an MHC monomer and not
present in dissociated components of the MHC complex. For example,
for a class I MHC monomer complex, the monoclonal antibody can bind
to a conformational epitope present in beta-2 microglobulin only
when incorporated into the ternary complex. Alternatively, the
monoclonal antibody can recognize a conformational epitope present
in the MHC monomer or modified MHC monomer being used in a
particular invention system or method. The monoclonal antibody may
be species-matched to the MHC monomers, for example, when the solid
support has attached HLA class I monomers, the monoclonal antibody
is a murine, human or humanized anti-MHC class I monoclonal
antibody. However, when the modified MHC monomer is a chimera
containing domains from more than one species, the anti-MHC
monoclonal antibody can be selected to bind to an epitope, (e.g., a
conformational epitope) present in only one of the domains. For
example, as illustrated in FIG. 9, a ternary complex containing
modified MHC monomer that is a chimera containing alpha-1 and alpha
2 domains of HLA-A2 heavy chain and a murine alpha-3 domain of H-2
Kb can be detected by a murine monoclonal antibody that binds to a
conformational domain in the murine alpha-3 domain.
[0090] When the MHC monomer is an HLA monomer, the monoclonal
antibody can be any anti-MHC class I monoclonal antibody that
recognizes any subclass of HLA monomer in a ternary complex, i.e.,
HLA-A, HLA-B or HLA-C. A preferred anti-MHC-class I monoclonal
antibody for use in the invention systems and methods is a mouse
IgG2a conformation dependent anti-HLA monoclonal antibody produced
by hybridoma B9.12.1, which as been deposited under the provisions
of the Budapest Treaty on the International Recognition of the
Deposit of Microorganisms for the Purpose of Patent Procedure and
the Regulations thereunder (Budapest Treaty) at Collection
Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur
25, Rue du Docteur Roux, F75724 Paris Cedex 15 France, under
registration number CNCM I-2941. This assures maintenance of viable
cultures for 30 years from the date of deposit. The organisms will
be made publicly available by CNCM under the terms of the Budapest
Treaty and assures permanent and unrestricted availability of the
progeny of the culture to the public upon issuance of the pertinent
U.S. patent or upon laying open to the public of any U.S. or
foreign patent application, whichever comes first, and assures
availability of the progeny to one determined by the U.S.
Commissioner of Patents and Trademarks to be entitled thereto
according to 35 U.S.C. .sctn. 122 and the Commissioner's rules
pursuant thereto (including 37 CFR .sctn. 1.14 with particular
reference to 886 OG 638).
[0091] In one embodiment, the monoclonal antibody used in the
invention systems and methods is provided with a detectable label,
i.e., a label that produces a detectable signal as is known in the
art. Labels may be conjugated to the antibody using any of a
variety of procedures known in the art. Alternatively, the antibody
can be produced to include a label, such as a radioactive amino
acid. Labels suitable for use in the invention systems, kits and
methods include, but are not limited to, radioisotopes,
fluorochromes, enzymes, biotin and electron dense molecules.
Binding of the monoclonal antibody indicates formation of a ternary
complex by binding of an MHC-binding peptide to the monomer and can
be easily detected and/or quantified by detecting the signals
produced by the signal entity after washing away unbound antibody
and other components of the system. A detectable label presently
preferred is a fluorescent label, e.g., FITC. The binding of
fluorescently labeled antibodies on the solid support can be
readily detected using a fluorometer or by fluorescence determined
flow cytometry.
[0092] As an alternative to use of a monoclonal antibody to
determine binding of the monomer to a suitable MHC binding peptide,
those of skill in the art will understand that any method that
separates complexes based on weight or electronic properties, such
as gel separations or electrophoresis, can be used to determine
whether a class I or class II monomer binds an MHC-binding
peptide.
[0093] The invention system can be provided either as part of
another system or as a kit. For example, microtiter plates coated
with the MHC monomers or modified monomers, e.g., in dried form,
can be provided in a kit, which can optionally additionally
include, in separate vials or containers, an anti-MHC monoclonal
antibody or an anti-beta-2 microglobulin antibody, as described
herein, and a control peptide that binds specifically to the
monomers attached to the solid support. In one embodiment, the kit
includes an instruction explaining the procedures for using the
system to conduct immunoassays, e.g., the methods provided by the
present invention. The kit can optionally also include any or all
of the following: denaturing or refolding buffers, controls for the
MHC monomers, the peptide, or the monoclonal antibody.
[0094] In yet another embodiment, the invention provides methods
for determining binding between a MHC monomer or modified MHC
monomer and a putative MHC-binding peptide to be tested for binding
to the monomer(s). In this method for assaying binding of a
putative MHC-binding peptide, a solid surface having attached
thereto a plurality of MHC monomers or modified MHC monomers is
incubated in the presence and absence of the putative MHC-binding
peptide. Preferably the solid surface is one belonging to an
invention system or kit and is prepared as described herein. If the
MHC monomers attached to the solid support at the start of the
assay procedure are in a reconstituted form, the MHC monomers are
prepared for the assay by exposure to denaturing conditions as
described herein, for example by exposure to a pH in the range from
about 2 to about 4, or exposure overnight to a temperature higher
than about 37.degree. C. After denaturation, unbound MHC-binding
peptides are washed away.
[0095] For the assay, the solid support with attached denatured MHC
monomers or modified MHC monomers is incubated with a putative
MHC-binding peptide under reconstituting conditions for a suitable
period of time to allow for formation of complexes. The
reconstituting conditions may also include a temperature in the
range from about minus 18.degree. C. to about 37.degree. C., for
example about 4.degree. C. to about 8.degree. C. For MHC class I
monomers, the reconstituting conditions will include the presence
of a sufficient amount of beta-2 microglobulin (or beta 2
microglobulin modified to increase binding or stabilize ternary
complex formation) to saturate the MHC monomers. For example, it is
contemplated that the beta 2-microglobulin may be modified by
attachment thereto of a stabilizing molecule, such as a leucine
zipper, or the like, to stabilize ternary complex formation.
Incubation with the putative MHC-binding peptide and beta-2
microglobulin will typically be required from about 12 hours or
overnight to about 48 hours to allow for complex formation.
[0096] After the reconstituting incubation, binding to the MHC
monomers of the putative MHC-binding peptide is determined by
contacting the MHC monomers on the solid support with a monoclonal
antibody that binds to a conformational epitope present only in the
MHC complex, for example a conformational epitope present in the
refolded MHC monomer of the class I ternary complex and not present
in a denatured MHC monomer. Binding of the antibody with the MHC
complex attached to the solid support indicates that the putative
MHC-binding peptide is an MHC-binding peptide specific for the MHC
monomers or modified MHC monomers used in the assay. For purposes
of comparison of the binding of the putative MHC-binding peptide to
that of a standard MHC-binding peptide, a parallel assay (e.g.,
under the same reconstituting conditions, same monomer, and in the
presence of the same monoclonal antibody) may be conducted using
the monomers. Binding of the monoclonal antibody in the parallel
assay to the ternary complex containing the standard MHC-binding
peptide can be compared to binding of the monoclonal antibody to
the corresponding complex in the test assay to aid in determining
the binding efficiency of the putative MHC-binding peptide, using
computational methods known in the art.
[0097] In still another embodiment, the invention provides methods
for determining the degree of binding affinity of an MHC monomer or
modified MHC monomer for a putative MHC-binding peptide. In this
embodiment, at least one denatured MHC monomer or modified MHC
monomer attached to a solid surface is incubated under
reconstituting conditions with the putative MHC-binding peptide and
a monoclonal antibody that specifically binds to a conformational
epitope created by formation of a complex containing a
corresponding reconstituted MHC monomer that is not present in any
of the dissociated components of the MHC complex. Binding of the
monoclonal antibody to the MHC complex so formed is compared with
binding of the monoclonal antibody to a corresponding complex
containing the same MHC monomer or modified MHC monomer and a known
MHC-binding peptide. The difference in the binding indicates the
relative degree of binding affinity of the reconstituted MHC
monomer or modified MHC monomer for the putative MHC-binding
peptide. For reconstitution of an MHC class I monomer, a suitable
amount of beta-2 microglobulin for complex formation of the total
amount of monomer in the assay must also be present For the
determination of the binding affinity of a peptide the test is done
in multiples using different peptide concentrations in each
parallel test. In practice of the invention methods, the MHC
monomers may belong to any species for which determination of
appropriate class I binding peptides is desired, including, without
limitation, murine and human or a chimera containing monomer
subunits from a combination of species or subtypes.
[0098] Various readily available means can be used to determine the
specific binding of the monoclonal antibody to the MHC complex
containing the reconstituted MHC monomer. For example, the binding
can be detected by directly labeling the monoclonal antibody with a
detectable label, i.e., one that produces a detectable signal, and
detecting the signal or via a secondary antibody that is detectably
labeled and recognizes the monoclonal antibody that binds to the
MHC complex containing the MHC monomer used in the assay. Suitable
detectable labels that can be used for this purpose are well known
in the art and include labels selected from the group consisting of
radioisotopes, fluorochromes, enzymes, biotin, electron dense
molecules, and the like. Fluorochromes or fluorescent labels are
currently preferred since binding can readily be detected by
subjecting the solid support to a fluorometer. For example, when
the solid support is a plate, such as a 96 well microtiter plate,
or beads, such as agarose A or agarose G beads, the assay can take
advantage of high through-put florescence scanning using any of the
methods known in the art.
[0099] The following examples are intended to illustrate but not to
limit the invention in any manner, shape, or form, either
explicitly or implicitly. While they are typical of those that
might be used, other procedures, methodologies, or techniques known
to those skilled in the art may alternatively be used.
EXAMPLE 1
[0100] Detection of Correctly Folded HLA Heavy Chain Monomers
[0101] This experiment demonstrates that MHC monomers when attached
to a solid support can be reconstituted so as to form a ternary
complex and be recognized and specifically bound by a
conformation-dependent anti-MHC monoclonal antibody. In other
words, MHC monomers bound to a solid support will correctly fold to
bind MHC-binding peptides. Table 1 below summarizes the major steps
for detecting the correctly folded HLA monomers upon peptide
binding. (See also FIG. 1.)
1TABLE 1 Step 1 Step 2 Step 3 Incubation of HLA After washing,
Washing and read out heavy chain coated incubation with in the
fluorometer plates with low pH different concentrations solution.
of peptide and a Washing constant concentration of beta-2
microglobulin and anti-HLA-class I- FITC mAb. Incubation time:
overnight or 24 h
EXAMPLE 2
[0102] Calibration of Anti-HLA-FITC Antibody
[0103] In this example BSA-Biotin-Avidin coated 96-well microtiter
plates were prepared used for a fluorimetric assay. HLA-A2m monomer
in ternary complex with binding peptide Mart-1 26-35L was incubated
at various concentrations with an anti-HLA-ABC-FITC or
anti-HLA-FITC monoclonal antibody at concentrations of 0, 0.25,
0.5, 1, 2, and 4 .mu.g/ml. Specifically, for each antibody
concentration, the HLA monomer was added at concentrations of 0,
0.0078, 0.0156, 0.03125, 0.0625, 0.125, 0.25, and 0.5 .mu.g/ml.
[0104] In this experiment the HLA heavy chain and the anti-HLA-FITC
antibody were incubated together for 40 min at room temperature
under shaking. The total fluorescence was read before washing the
plates to remove unbound antibody. Then, plates were washed three
times to remove any unbound antibody, and the fluorescence of the
bound monomers was read.
[0105] As shown in FIG. 2, saturation occurred when the antibody
concentration reached 0.25 and 0.5 .mu.g/ml. However, the
fluorescence signal increased when the antibody was added at 1, 2
and 4 .mu.g/ml. This observation indicates that the antibody binds
two MHC monomers when added at 0.5 and 0.25 .mu.g/ml. In contrast,
upon incubation at 1, 2 or 4 .mu.g/ml, the antibody binds only one
HLA monomer. This explains the signal increase, e.g., 300
Fluorescence units (FU) with 0.5 .mu.g/ml of antibody and 0.5
.mu.g/ml of HLA heavy chain as compared with 600 FU with 1 .mu.g/ml
of antibody and 0.5 .mu.g/ml of HLA heavy chain. Another
observation was that between the concentrations of 2 and 4 .mu.g/ml
of antibody the signal remains constant. It was determined,
therefore, that 2 .mu.g/ml of anti-HLA-FITC mAb was an appropriate
saturation concentration to use for the assay.
EXAMPLE 3
[0106] Specificity of Anti-HLA-FITC Monoclonal Antibody.
[0107] A. Conformational Specificity
[0108] Experiments were designed to determine if the signal
produced from anti-HLA-class I-FITC antibody differs as a function
of the degree of dissociation of a stressed HLA monomer. The
particular HLA monomer used for the experiments was HLA heavy chain
monomer HLA-A*0201 containing binding peptide Mart1 27-35 in
ternary complex.
[0109] Different solutions containing the ternary complex at a
concentration of 10 jig/ml were prepared and incubated overnight at
the temperatures of 37.degree. C., 30.degree. C., 25.degree. C., or
4-8.degree. C. Antibody binding experiments as described above in
Example 2 were carried out using 2 .mu.g/ml of anti-HLA-FITC
conjugate to detectably label the HLA monomers remaining in ternary
complex attached to the solid support. A solution containing a
ternary complex of HLA monomer and Mart1 27-35 at a concentration
of 640 .mu.g/ml were incubated at -18.degree. C. as a control. A
solution, from a sample stored at -18.degree. C. at the
concentration of 640 .mu.g/ml, containing a ternary complex of
HLA-monomer and Mart-1 27-35 was diluted at the same concentration
than the other samples and included as control.
[0110] As shown in FIG. 3, it was found that incubation of the
ternary complex bound to the solid support at highest temperature
gave the weakest fluorescent signal, indicating that the ternary
complex of HLA heavy chain monomer gradually dissociated as the
temperature was increased. At one point, the anti-HLA-FITC
conjugate could no longer recognize the HLA monomer because of the
degree of dissociation of the ternary complex dissociation and the
fluorescence signal diminished accordingly, indicating that the
anti-HLA-FITC conjugate specifically recognizes correctly folded
reconstituted HLA monomers, but not denatured monomers.
[0111] B. Heavy Chain Monomer Specificity
[0112] As shown in Table 2, different HLA heavy chain monomers for
human alleles, A2, A3, A11, B7, B8, as well as one mouse allele Kd
were incubated with the anti-HLA-class I-FITC antibody.
2 TABLE 2 Human alleles Mouse allele HLA-A*0201/Mart1 2635L
H-2Kd/Flu HLA-A*0301/EBV HLA-A*1101/EBV HLA-B*0702/gp41
HLA-B*0801/Nef
[0113] The antibody binding experiments were carried out as
described above. The concentration of the anti-HLA-class I-FITC
antibody used was 2 .mu.g/ml.
[0114] As shown in FIG. 4, all the reconstituted HLA-A and -B
monomers were detectable with the anti-HLA-class I monoclonal
antibody. As expected, no signal was detected when a ternary
complex containing the mouse allele (H-2Kd/Flu) was attached to the
plate, confirming the specificity of the antibody to human HLA.
Variations of signal between different alleles were likely due to
concentration precision and storage conditions of the HLA monomers,
e.g., freeze, thaw, etc.
[0115] Coating of MHC Heavy Chain Monomers to Plates, Plate Storage
and Reconstitution
[0116] Biotinylated MHC monomers in a ternary complex with Mart1
27-35 peptide at the concentration of 5 .mu.g/ml were attached to
avidin coated plates. After saturating the plates with a
sugar-containing buffer overnight at 4.degree. C. to 8.degree. C.,
the plates were dried overnight at 30.degree. C. and 19 % humidity.
After the plates were dried under these conditions, it was found
that the HLA monomers were dissociated from the ternary complex.
Therefore, it was not necessary to strip the MHC-binding peptide
from the monomers with low pH in preparation for use of the plates
in the binding assay.
[0117] For the antibody binding assay, 10 nM to 100 .mu.M of HBc
high affinity peptide (the affinity can be calculated as
1.8.times.10.sup.-7M) were incubated with 10 .mu.g/ml of .beta.2
microglobulin and the monomer-coated plates were incubated with one
of the three different buffers containing ingredients as described
below:
[0118] Buffer 1: Tris, Arginine, EDTA, GSH, GSSG and BSA
[0119] Buffer 2: Tris, NaCl, EDTA, NaN.sub.3, BSA and 0.05% TWEEN
20.RTM. detergent
[0120] Buffer 3: Tris, NaCl, EDTA, NaN.sub.3, BSA and 0.05%
NONIDET.RTM. P40 detergent.
[0121] It was found that peptide binding and reconstitution of the
monomers occurred at 2 temperatures: 4.degree. C. -8.degree. C. and
room temperature.
[0122] Renaturation of the HLA monomers was tested after 24 hours
and 48 hours of incubation with 2 .mu.g/ml of anti-HLA-class I-FITC
conjugate. As shown in FIG. 5, the FITC signal increased as a
function of the peptide concentration. This result shows that the
HLA monomer renaturated by incorporation into a ternary complex and
that renaturation of MHC monomers can be effectively detected with
an anti-HLA-class I-FITC antibody. It was found that the best
renaturation buffer was the Buffer 2 containing TWEEN 20.RTM..
Interestingly no refolding was measured with Buffer 1.
[0123] Under the conditions tested here, the best temperature for
the antibody binding assay was 4.degree. C. -8.degree. C. and the
best incubation period to allow renaturation was 24 hours.
[0124] Material and Methods
[0125] A. Reagents.
[0126] Fine chemicals, unless otherwise stated, were from Merck
(Darmstadt, Germany) and Carlo Erba (Rodeno, Italy). Biotinylated
BSA as well as avidin was obtained from Immunotech (Marseille,
France). LUMITRAC-600 White 96-well microtiter plates were from
Greiner [PN: 655074 LUMITRAC 600; (Frickenhausen, Germany). SA-PE
as well as HLA-A*0301/EBV HLA heavy chain were from Immunomics (
(San Diego, Calif.). Anti-HLA-class I monoclonal antibody
conjugated to FITC (clone: B9.12.1) was from Antibody Manufacturing
Service of Immunotech. Part Number: IM1838. This antibody is a
mouse 1 gG2a monoclonal antibody.
[0127] B. Preparation of Avidin Coated 96-well Microtiter
Plates.
[0128] Each well of white 96-well microtiter plates were coated
with 200 .mu.l of a 5 .mu.g/ml biotinylated BSA solution in PBS and
the plates were incubated for 16 hours at 4.degree. C. The plates
were washed and then 200 .mu.l/well of avidin solution at 5
.mu.g/ml was added. The plates were then incubated for another 16
hours at 4.degree. C. Subsequently the plates were washed and a
blocking, drying solution was added. The plates were incubated
again for another 16 hours. Afterwards, the solution was poured off
and the plates were slapped face down on paper towels. Then the
plates were placed in a special drying room for 24 hours.
Afterwards the plates were placed individually in a self-locking
bag until use.
[0129] C. Monomer Immunoassay Procedure.
[0130] The assay procedure was as follows. Each sample 200
.mu.l/well containing the HLA monomer in ternary complex at 0.25
.mu.g/ml and diluted in Tris 10 mM, NaCl 150 mM, EDTA 0.5 mM, NaN3
0.1%, BSA 0.2%, was loaded into wells of the avidin-coated plate
and incubated for 1 hour at room temperature on an orbital shaker
in the dark. The wells were then rinsed three times with an
automatic washer (SLT, Salzburg, Austria) using 300 .mu.l of a 9
g/l NaCl solution containing 0.05% TWEEN 80.RTM.. Subsequently 200
.mu.l/well of FITC-conjugated anti-HLA-class I antibody at 2
.mu.g/ml were added. The plates were incubated for 45 min at room
temperature on an orbital shaker in the dark, washed three times,
and 200 .mu.l/well of Tris 10 mM, NaCl 150 mM, EDTA 0.5 mM, NaN3
0.1%, BSA 0.2% were added. The FITC fluorescence was measured with
a Perkin Elmer LS-50 B fluorometer following these parameters:
[0131] Excitation=405 nm
[0132] Emission=525 nm
[0133] Emission filter=515 nm
[0134] Band pass (Exc,Emi)=5.15 nm
[0135] 0.5 sec/well
[0136] The assay procedure is further summarized in Table 3
below.
3TABLE 3 Step 1 Step 2 Step 3 Step 4 Mix HLA heavy Incubate 200
.mu.l/well Three washes Three washes chain and of each sample in
the Add 200 .mu.l/well of Add 200 .mu.l of buffer streptavidin PE
96-well streptavidin anti-HLA-class I mAb Fluorescence coated white
plates. at 2 .mu.g/ml determination Incubate 1 hour at Incubate 45
min at room temperature in room temperature in the dark under the
dark under agitation agitation
[0137] Calibration of the Anti-HLA-Class I-FITC Antibody
[0138] HLA-A*0201/Mart1 reconstituted monomers in various
concentrations was incubated with various concentrations of the
anti-HLA-class I-FITC mAb. As shown in FIG. 6, a plateau was
reached with concentrations of anti-HLA-class I mAb at 1 to 2
.mu.g/ml for all HLA heavy chain monomer concentrations.
[0139] A dose response curve at various concentrations of
reconstituted monomers was plotted using 2 .mu.g/ml of anti-HLA
-ABC mAb. As shown in FIGS. 7A and 7B, the signal remained linear
with increasing concentrations until 0.5 .mu.g/ml of reconstituted
HLA monomer was used. Concentrations of the reconstituted HLA
monomer higher than 0.5 .mu.g/ml provided signals that were very
close to a plateau. The data summarized in FIGS. 7A and 7B
demonstrate that for the best result, the assay conditions should
include 0.25 .mu.g/ml of reconstituted HLA monomer and 2 .mu.g/ml
of anti-HLA-class I FITC mAb. These data also indicated that the
sensitivity of the assay is about 4 to 6 ng/ml of the reconstituted
HLA monomer.
[0140] Specificity
[0141] The specificity of the anti-HLA-class I-FITC antibody for
HLA monomer in ternary complex was tested against different human
alleles. A dose response curve was prepared as described above for
each of the following HLA monomer/peptide ternary complexes.
[0142] HLA-A*0201/Mart1 2635L
[0143] HLA-A*0301/EBV
[0144] HLA-B*0702/HIV
[0145] HLA-B*0801/HIV
[0146] HLA-A-*1101/EBV
[0147] H-2Db/HA1
[0148] As shown in FIG. 8, all the human alleles were recognized
very well by the same anti-HLA-class I-FITC antibody. No signal was
obtained when a human allele was replaced by a mouse allele,
indicating that the antibody used is specific for human class I
alleles and should be used only in assays involving human alleles.
A conformational anti-mouse H-2 antibody was found suitable for use
in the assays involving mouse HLA monomers.
EXAMPLE 4
[0149] Measurement Of The Peptide-MHC Off Rate
[0150] For effective CD8+ T cell responses, class I MHC molecules
must bind many peptides of diverse sequence in sufficient abundance
for a long period of time. Many tumor cells appear to escape the
immune response because antigenic peptides do not bind well to
class I MHC molecules that present them. If a peptide does not bind
efficiently to the MHC molecule, circulating T cells will not
recognize the MHC ternary complex, and cells presenting them will
not be eliminated.
[0151] Typical half-lives of immunodominant peptides are greater
than 20 hours at 37.degree. C. (Stuber, et al., (1994) Eur. J.
Immunol. 24, 765-768, and Pogue, et al., (1995) Proc. Natl. Acad.
Sci. US 92, 8166-8170). From this evidence, a test was developed to
use the invention solid phase assay to determine the stability of
various complexes at different temperatures, and thus calculate the
off rate of the peptides. This parameter is very valuable to know
when peptides are used in vaccination for the purpose of eliciting
an immune response.
[0152] Measurement of the peptide off rate: Monomer
HLA-A*0201/Mart-1 2635L was loaded in four different 96-well avidin
coated plates. The plate was incubated for two hours under shaking
at room temperature. After washing and stripping with citrate
phosphate buffer at pH 3.2 the monomer was reconstituted with high
affinity peptides HbV core, Mart-1 2635L, with intermediate
affinity peptide Mart-1 26-35 as well as the low affinity peptide
Mart-1 27-35. Free beta2 microglobulin as well as the
anti-HLA-ABC-FITC monoclonal antibody was added at the same time
with the peptide. The plates were incubated at 21.degree. C. under
shaking overnight. After that, the plates were washed and the level
of the fluorescence determined. After this Tris buffer containing
the BSA was added to each well and the plates were re-incubated at
different temperatures, one plate was incubated at 4.degree. C.,
one at 21.degree. C., one at 32.5.degree. C. and the last one at
37.degree. C., respectively. Some strips of each plate were washed
at different times--4 hours, 24 hours and 48 hours--and the
fluorescence at different times was determined.
[0153] B0 is the fluorescence determined at time zero. The time
zero corresponds to the moment when the plates were washed once the
monomer was reconstituted and the plates were placed at different
temperatures. B is the fluorescence obtained at each time. After
the N1 (Fluorescence Emission) as a function of the time was
plotted. Linear regression was calculated and the Half-life was
calculated as T1/2=0.69/slope of the curves. Alternatively, the
data can be fitted equally well using non-linear regression
applying a one phase exponential decay and a plateau equal to
zero.
[0154] Results of these assays are shown in Table 4 below:
4 TABLE 4 T.sub.1/2 hours Peptide 4.degree. C. 21.degree. C.
32.5.degree. C. 37.degree. C. HBVcore 13800 493 101 21.5 2635L
>1725 345 98.6 22.4 2635 186 20.3 2.5 1 2735 56.1 8.8 1.3
0.96
[0155] It was observed that high affinity peptides, such as HBV
core and Mart-1 27-35 had a very good stability at 37.degree. C.
and 32.5.degree. C. In contrast, peptide Mart-1 26-35 as well
peptide Mart-1 27-35 showed a very high off rate at 37.degree. C.
Differences were found also when complexes were incubated at
21.degree. C. These results indicate that the assay can be used to
determine the off rate of peptides from the MHC ternary complex
(see FIG. 10; FIGS. 10A-D show graphs of the dissociation curves
for renatured peptides. FIGS. 10E-H shows graphs of the off rates
for peptides. FIG. 10I shows the effect of temperature on monomer
dissociation).
[0156] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
* * * * *